Methods of treating cancer with interferon wherein the cancer cells are hla negative or have reduced hla expression

ABSTRACT

The invention described herein relates to methods for treating a patient having a plurality of HLA-negative cancer cells or cancer cells with reduced HLA expression with IFN-alpha in an amount sufficient to expand and/or activate immune cells such that the activated and/or expanded immune cells kill one or more of HLA-negative cancer cells or the cancer cells with reduced HLA expression.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Patent Application No.16/068,392 filed on Jul. 6, 2018, which is a U.S. National Stage Filingunder 35 U.S.C. § 371 of International Patent Application Serial No.PCT/US2017/012580 filed Jan. 6, 2017, which claims priority to U.S.Provisional Patent Application No. 62/276,198 filed Jan. 7, 2016;priority to each of which is claimed, and each of which is herebyincorporated by reference herein in its entirety.

FIELD

This disclosure relates to novel methods for treating cancer in apatient having a plurality of Human Leukocyte Antigen (“HLA”)-negativecancer cells or cancer cells with reduced HLA expression by usingInterferon-alpha in an amount sufficient to expand and/or activateimmune cells, e.g., mature killer cells, to kill the HLA-negative cancercells or the cancer cells with reduced HLA expression.

BACKGROUND

The therapeutic efficacy of conventional cancer treatments, includingchemotherapy, surgery, and radiotherapy, are limited for a large numberof cancers. For example, chemotherapeutic agents are generallyconsidered ineffective in treating brain metastases from solid tumorsbecause the drugs cannot penetrate the intact blood brain barrier.Moreover, chemotherapeutic agents may cause considerable side effects onhealthy cells in patients. Allen T M. (2002) Cancer 2:750-763. Further,cancer recurrence in cancer patients is often associated with surgeryand radiotherapy, which can lead to higher mortalities in cancerpatients. Clarke M, et al. (2005) Lancet 366 (9503):2087-106.

Cancer immunotherapy has emerged as an important therapy for cancers,particularly for advanced and refractory cancers. Compared to theconventional therapies, immunotherapy demonstrates great potential forcancer treatment with significantly higher specificity and efficacy.Since immunotherapy uses the patients' own immune system to destroycancer cells, it has little or no side effects that are often associatedwith traditional treatment. The use of genetically modified T-cells forcancer treatment is one of the most noted cancer immunotherapies.Couzin-Frankel J. (2013) Science 20342(6165):1432-3. Activatedcancer-specific T-cells can kill cancer cells by recognizing thespecific antigens or peptides expressed on the cells.

Therefore, there remains a need in the art to develop a novel cancerimmunotherapy with improved efficacy.

SUMMARY

A drawback of certain T-cell based cancer therapies is that cancer cellscan develop escape mechanisms to evade the course of immunotherapy,particularly the immunotherapy directed by cancer-specific T-cells.Embodiments of the technology herein generally is predicated, at leastin part, on the discovery that a patient having a plurality ofHLA-negative cancer cells or cancer cells with reduced HLA expressioncan be effectively treated with interferon-alpha (“IFN-alpha”) in anamount sufficient to expand and/or activate immune cells such that theactivated and/or expanded immune cells kill one or more of HLA-negativecancer cells or the cancer cells with reduced HLA expression. In oneembodiment, the immune cells can be T-cells, B-cells, or NK cells. Inanother embodiment, the patient is HLA-negative or has reducedexpression of a class I HLA, a class II HLA, or a class III HLA.Preferably, the HLA is class I HLA or class II HLA.

In one aspect of the invention, IFN-alpha can be one or more ofIFN-alpha 1, IFN-alpha 2, IFN-alpha 4, IFN-alpha 5, IFN-alpha 6,IFN-alpha 7, IFN-alpha 8, IFN-alpha 10, IFN-alpha 14, IFN-alpha 16,IFN-alpha 17, or IFN-alpha 21.

Preferably, IFN-alpha is IFN-alpha 2, IFN-alpha 8, or IFN-alpha 10. Inother preferred embodiments, the IFN-alpha is IFN-alpha 8. In oneembodiment, IFN-alpha is a synthetic or recombinant IFN-alpha.

In one aspect of the invention, the effective amount of the IFN-alpha isa sub-therapeutic amount of the IFN-alpha. It is contemplated thatadministration of a sub-therapeutic amount of IFN-alpha can stillactivate, or expand the immune cells, including NK cells, such that theactivated and/or expanded immune cells can kill one or more ofHLA-negative cancer cells or cancer cells with reduced HLA expression.In one embodiment, the amount of IFN-alpha is sub-therapeutic comparedto an amount of IFN-alpha that is used to treat a cancer. In oneembodiment, the sub-therapeutic amount of IFN-alpha can be about 1%,about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about35%, about 40%, about 45%, about 50%, about 55%, or about 60% of atherapeutic dosage of the IFN-alpha. In another embodiment, theIFN-alpha is provided in an amount ranging from about 5×10¹ units persquare meter of body surface (U/m²) to about 2×10⁵ U/m2, or any subvalue or sub range therein. Administration of a sub-therapeutic amountof IFN-alpha allows for greater targeting of the immune cells (e.g., NKcells) to the tumor, or decreasing any potential side effects associatedwith IFN-alpha, or both.

In one embodiment, to expand, activate, and/or stimulate the immunecells, the IFN-alpha can be administered orally, via vein injection,muscle injection, peritoneal injection, subcutaneous injection, nasal ormucosal administration, or by inhalation via an inspiratory.

In one aspect of the invention, the cancer comprises a population ofcancer cells that initially express an HLA prior to comprising apopulation that is HLA-negative or have a reduced HLA expression. In oneembodiment, the patient has received or is selected based upon havingreceived an immunotherapy for the cancer. In one embodiment, theimmunotherapy is a T-cell therapy.

In one embodiment, the IFN-alpha is administered as part of acombination therapy. In another embodiment, the combination therapycomprises one or more of chemotherapy, radiotherapy, and immunotherapy.

One aspect of the invention relates to a method for treating a cancer,which comprises administering an immune cell to a subject in needthereof, wherein said immune cell is activated by IFN-alpha, furtherwherein the cancer has no or reduced expression of a HLA compared to anormal control. In one embodiment, the immune cell is a T-cell, aB-cell, or a NK-cell. Preferably, the immune cell is a NK cell. In oneembodiment, the NK cell is a NK cell line, e.g., a NK-92 cell.

In one embodiment, the HLA is a class I HLA antigen, a class II HLAantigen, or a class III HLA antigen. Preferably, the HLA is a class IHLA antigen or a class II HLA antigen.

In another embodiment, the source of the immune cells is autologous,allogeneic, or xenographic.

In one embodiment, the immune cells are administered intravenously,intraperitoneally, intramuscularly, or subcutaneously.

In one embodiment, the immune cells, such as NK cells, are activated byincubating the immune cells with the IFN-alpha ex vivo. In anotherembodiment, the immune cells are incubated ex vivo with the IFN-alpha ata concentration from 5×10¹ U/1×10⁶ cells to 1×10⁴U/1×10⁶ cells.

In one embodiment, the immune cells, such as NK cells, are administeredin combination with an anti-tumor agent. In another embodiment, theanti-tumor agent is a chemotherapy agent, a radiotherapy agent, or animmunotherapy agent, such as an anti-cancer vaccine, an anti-cancerantibody, or an immune checkpoint inhibitor.

In one embodiment, the immune cell is modified to express a tumor cellhoming receptor on the outer cell surface of the immune cell. In anotherembodiment, the tumor cell homing receptor is a chimeric antigenreceptor, an Fe receptor, or combinations thereof.

In one embodiment, the patient or subject is human.

In one embodiment is provided a composition comprising immune cells andan effective amount of interferon alpha (IFN-alpha) to activate theimmune cells.

In one embodiment, the IFN-alpha comprises one or more of IFN-alpha 1,IFN-alpha 2, IFN-alpha 4, IFN-alpha 5, IFN-alpha 6, IFN-alpha 7,IFN-alpha 8, IFN-alpha 10, IFN-alpha 14, IFN-alpha 16, IFN-alpha 17, orIFN-alpha 21.

In one embodiment, the immune cells are NK cells. In one embodiment, theNK cells are NK-92 cells.

In one embodiment, the composition comprises IFN-alpha at aconcentration from 5×101 U/1×106 cells to 1×104 U/1×106 cells.

In one embodiment, the composition further comprises a pharmaceuticallyacceptable excipient.

DETAILED DESCRIPTION

After reading this description, it will become apparent to one skilledin the art how to implement the invention in various alternativeembodiments and alternative applications. However, not all embodimentsof the present invention are described herein. It will be understoodthat the embodiments presented here are presented by way of an exampleonly, and are not limited. As such, this detailed description of variousalternative embodiments should not be construed to limit the scope orbreadth of the present invention as set forth below.

Before the present invention is disclosed and described, it is to beunderstood that the aspects described below are not limited to specificcompositions, methods of preparing such compositions, or uses thereof assuch may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularaspects only and is not intended to be limiting.

Throughout this disclosure, various publications, patents and publishedpatent specifications are referenced by an identifying citation. Thedisclosures of these publications, patents and published patentspecifications are hereby incorporated by reference in their entiretyinto the present disclosure.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

In this specification and in the claims that follow, reference will bemade to a number of terms that shall be defined to have the followingmeanings:

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise.

All numerical designations, e.g., pH, temperature, time, concentration,amounts, and molecular weight, including ranges, are approximationswhich are varied (+) or (−) by 10%, 1%, or 0.1%, as appropriate. It isto be understood, although not always explicitly stated, that allnumerical designations may be preceded by the term “about.” It is alsoto be understood, although not always explicitly stated, that thereagents described herein are merely examples and that equivalents ofsuch are known in the art.

“Optional” or “optionally” means that the subsequently described eventor circumstance can or cannot occur, and that the description includesinstances where the event or circumstance occurs and instances where itdoes not.

The term “about” when used before a numerical designation, e.g.,temperature, time, amount, concentration, and such other, including arange, indicates approximations which may vary by (+) or (−) 10%, 5%,1%, or any subrange or sub value there between.

Preferably, the term “about” when used with regard to a dose amountmeans that the dose may vary by +/−10%.

The term “comprising” or “comprises” is intended to mean that thecompositions and methods include the recited elements, but not excludingothers. “Consisting essentially of” when used to define compositions andmethods, shall mean excluding other elements of any essentialsignificance to the combination. For example, a composition consistingessentially of the elements as defined herein would not exclude otherelements that do not materially affect the basic and novelcharacteristic(s) of the claimed invention. “Consisting of” shall meanexcluding more than trace amount of other ingredients and substantialmethod steps recited. Embodiments defined by each of these transitionterms are within the scope of this invention.

The terms “patient,” “subject,” “individual,” and the like are usedinterchangeably herein, and refer to any animal, or cells thereofwhether in vitro or in situ, amenable to the methods described herein.In a preferred embodiment, the patient, subject, or individual is amammal. In some embodiments, the mammal is a mouse, a rat, a guinea pig,a non-human primate, a dog, a cat, or a domesticated animal (e.g. horse,cow, pig, goat, sheep). In especially preferred embodiments, thepatient, subject or individual is a human.

The term “treating” or “treatment” covers the treatment of a disease ordisorder described herein, in a subject, such as a human, and includes:(i) inhibiting a disease or disorder, i.e., arresting its development;(ii) relieving a disease or disorder, i.e., causing regression of thedisease or disorder; (iii) slowing progression of the disease ordisorder; and/or (iv) inhibiting, relieving, or slowing progression ofone or more symptoms of the disease or disorder. For example, treatmentof a cancer or tumor includes, but is not limited to, reduction in sizeof the tumor, elimination of the tumor and/or metastases thereof,remission of the cancer, inhibition of metastasis of the tumor,reduction or elimination of at least one symptom of the cancer, and thelike.

The term “administering” or “administration” of an agent, drug, or animmune cell, including but not limited to a natural killer cell, to asubject includes any route of introducing or delivering to a subject acompound to perform its intended function. Administration can be carriedout by any suitable route, including orally, intranasally, parenterally(intravenously, intramuscularly, intraperitoneally, or subcutaneously),or topically. Administration includes self-administration and theadministration by another.

It is also to be appreciated that the various modes of treatment orprevention of medical diseases and conditions as described are intendedto mean “substantial,” which includes total but also less than totaltreatment or prevention, and wherein some biologically or medicallyrelevant result is achieved.

As used herein, the term “effective amount” refers to an amount ofIFN-alpha which is capable of activating the immune cells. The preciseeffective amount will vary, for example based on the type of the immunecells to be activated and the subtype of IFN-alpha administered.

As used herein, the term “sub-therapeutic amount” is used to describe anamount of IFN-alpha that is below the amount of IFN-alpha conventionallyused to treat a cancer. For example, a sub-therapeutic amount is anamount less than that defined by the manufacturer as being required fortherapy. For example, in some non-limiting embodiments, asub-therapeutic amount of IFN-alpha can be an amount less than 2×10⁷U/m², including any sub value or subrange below that amount and above5×10¹ U/m² Other non-limiting examples of a sub-therapeutic amountsinclude those described in Ningram, R A (2014) Scientifica Volume 2014,Article ID 970315, and references cited within, each of which isincorporated herein by reference in its entirety.

As used herein, the term “therapeutic amount” refers to an amount ofIFN-alpha conventionally used to treat a cancer. A “therapeutic amount”can be any amount defined by the manufacturer as being required fortherapy. The therapeutic amount of the IFN will vary depending on thetumor being treated and its severity as well as the age, weight, etc.,of the patient to be treated. For example, for hairy cell leukemia, thetherapeutic amount for IFN-alpha 2b is 2×10⁶ U/m²; for malignantmelanoma, the therapeutic amount is 2×10⁷ U/m². The skilled artisan willbe able to determine appropriate dosages depending on these and otherfactors. The compositions can also be administered in combination withone or more additional therapeutic compounds. In the methods describedherein, the therapeutic compounds may be administered to a subjecthaving one or more signs or symptoms of a disease or disorder.

As used herein, the term “interferon” or “IFN” means the family ofhighly homologous species-specific proteins that inhibit viralreplication and cellular proliferation and modulate immune response.

The terms “α-interferon,” “alpha interferon,” “interferon alpha” and“human leukocyte interferon” are used interchangeably in thisapplication to describe members of this group. Both naturally occurringand recombinant α-interferons, including consensus interferon, may beused in the practice of the invention.

As used to describe the present invention, “natural killer cells” or “NKcells” are cells of the immune system that kill target cells in theabsence of a specific antigenic stimulus, and without restrictionaccording to MHC class. Target cells may be tumor cells or cellsharboring viruses. NK cells are characterized by the presence of CD56and the absence of CD3 surface markers.

As used to describe the present invention, the terms “cytotoxic” and“cytolytic,” when used to describe the activity of effector cells suchas NK cells, are intended to be synonymous. In general, cytotoxicactivity relates to killing of target cells by any of a variety ofbiological, biochemical, or biophysical mechanisms. Cytolysis refersmore specifically to activity in which the effector lyses the plasmamembrane of the target cell, thereby destroying its physical integrity.This results in the killing of the target cell. Without wishing to bebound by theory, it is believed that the cytotoxic effect of NK cells isdue to cytolysis.

The term “ex vivo” as used herein means that in vitro expansion,activation, and/or stimulation of immune cells, including T-cells,B-cells, and NK cells, prior to introducing the expanded, activated,and/or stimulated immune cells to a subject or a patient.

The term “therapeutic” as used herein means a treatment and/orprophylaxis. A therapeutic effect is obtained by suppression, remission,or eradication of a disease state.

As used herein, “endogenous” refers to any material from or producedinside an organism, cell, tissue or system.

As used herein, the term “exogenous” refers to any material introducedfrom or produced outside an organism, cell, tissue or system.

The term “human leukocyte antigens” or “HLA,” refers to proteins(antigens) found on the surface of white blood cells and other tissuesthat are responsible for regulation of the immune system. HLA includesclass I, class II, and class III HLA molecules.

The term “kill” with respect to a cell/cell population is directed toinclude any type of manipulation that will lead to the death of thatcell/cell population.

“Antibodies” as used herein include polyclonal, monoclonal, singlechain, chimeric, humanized and human antibodies, prepared according toconventional methodology.

“Cytokine” is a generic term for non-antibody, soluble proteins whichare released from one cell subpopulation and which act as intercellularmediators, for example, in the generation or regulation of an immuneresponse. See Human Cytokines: Handbook for Basic & Clinical Research(Aggarwal, et al. eds., Blackwell Scientific, Boston, Mass. 1991) (whichis hereby incorporated by reference in its entirety for all purposes).

“Immune cells” as used herein are cells of hematopoietic origin that areinvolved in the specific recognition of antigens. Immune cells includeantigen presenting cells (APCs), such as dendritic cells or macrophages,B-cells, T-cells, natural killer cells, etc.

The term “anti-cancer therapy” as used herein refers to cancertreatments, including but not limited to, chemotherapy and radiotherapy,as well as immunotherapy and vaccine therapy.

As used herein, “chimeric antigen receptors” or “CARs” refer to fusionproteins comprised of an antigen recognition moiety and immune cellactivation domains. Eshhar et al., (1993) Proc. Natl. Acad. Sci., 90(2):720-724. For example, a CAR is an artificially constructed hybridprotein or polypeptide containing an antigen binding domain of anantibody (e.g., a single chain variable fragment (scFv)) linked toT-cell signaling or T-cell activation domains. CARs have the ability toredirect T-cell specificity and reactivity toward a selected target(i.e., a tumor cell) in a non-MHC-restricted manner, exploiting theantigen-binding properties of monoclonal antibodies. Thenon-MHC-restricted antigen recognition gives T-cells expressing CARs theability to recognize an antigen independent of antigen processing, thusbypassing a major mechanism of tumor escape. Moreover, when expressed inT-cells, CARs advantageously do not dimerize with endogenous T-cellreceptor (TCR) alpha and beta chains.

The terms “express” and “expression” mean allowing or causing theinformation in a gene or DNA sequence to become manifest, for exampleproducing a protein by activating the cellular functions involved intranscription and translation of a corresponding gene or DNA sequence. ADNA sequence is expressed in or by a cell to form an “expressionproduct” such as a protein (e.g., a CAR). The expression product itself,e.g. the resulting protein, may also be said to be “expressed.” Anexpression product can be characterized as intracellular, extracellularor secreted. The term “intracellular” means something that is inside acell. The term “extracellular” means something that is outside a cell. Asubstance is “secreted” by a cell if it appears in significant measureoutside the cell, from somewhere on or inside the cell.

The term “transfection” means the introduction of a foreign nucleic acidinto a cell. The term “transformation” means the introduction of a“foreign” (i.e. extrinsic or extracellular) gene, DNA or RNA sequence toan ES cell or pronucleus, so that the cell will express the introducedgene or sequence to produce a desired substance in a geneticallymodified animal.

The term “reduced expression” in reference to HLA expression means adecrease in HLA on the surface of a cancer cell. For example, reducedexpression can be about 1.5 times, or alternatively, about 2.0 times, oralternatively, about 2.5 times, or alternatively, about 3.0 times, oralternatively, about 5 times, or alternatively, about 10 times, oralternatively about 50 times, or yet further alternatively more thanabout 100 times lower expression than the expression level detected in acontrol sample (e.g. an earlier cancer biopsy, compared to expression ontypical cancer cells of the type, or on NK cells). The control samplealso can be cells collected from a person not having cancer.

Overview

The current invention is predicated, in part, on the surprisingdiscovery that treatment of a cancer expressing no or reduced HLAantigen with immune cells activated by IFN-alpha, either in vivo or exvivo, provides for unexpectedly improved therapeutic outcomes.

As will be apparent to the skilled artisan upon reading this disclosure,the present disclosure relates to methods for treating a patientsuffering from a cancer expressing no or reduced HLA antigen by treatingthe patient with immune cells, e.g., NK cells, which are activated by aneffective amount or a sub-therapeutic amount of IFN-alpha.

Immune Cells and Immunotherapy

Immune cells are part of the complex network that defends the bodyagainst pathogens and other foreign substances, including cancer cells.The cells of the immune system include B-cells, dendritic cells,granulocytes, innate lymphoid cells (ILCs), megakaryocytes,monocytes/macrophages, natural killer (NK) cells, and T-cells, amongothers. The innate immune response, which is carried out by phagocyticcells (e.g., macrophages and cytotoxic NK cells) is the first line ofdefense to pathogenic exposure. Subsequently, the adaptive immuneresponse includes antigen-specific defense mechanisms orchestrated byantigen-presenting cells (e.g., macrophages and dendritic cells).

Immunotherapy, including antibody and immune cell-based therapy, hasemerged as a standard treatments for a number of cancers. Adoptive celltransfer (“ACT”), being tested for the treatment of cancer and chronicinfections, has the potential to enhance antitumor immunity, augmentvaccine efficacy, and limit graft-versus-host disease. In ACT, immunecells from the patient are modified and engineered ex vivo to recognizeand attack the patient's own tumor. For adoptive T cell therapy, themodifications include altering the specificity of the T cell receptor(TCR) or introducing antibody-like recognition in chimeric antigenreceptors (CARs).

At present, however, while immunotherapy has demonstrated in numerousexperimental models as having considerable effectiveness, the clinicalresults have been less promising. One explanation is that tumors usemany strategies to evade the host immune response, includingdownregulation or weak immunogenicity of target antigens and creation ofan immune-suppressive tumor environment. T-cell mediated immunotherapyrequires that T-cells recognize and interact with a plurality of cellsurfaces molecules, including HLA, and peptides. The interaction ofT-cell and complexes of HLA/peptide is restricted, requiring a T-cellspecific for a particular combination of an HLA molecule and a peptide.If the specific complex is lacking (e.g., does not express) oneparticular HLA, there is no T-cell response even if the T-cell ispresent. This mechanism is involved in the immune system's response toforeign materials, in autoimmune pathologies, and in responses tocellular abnormalities. Studies have shown that tumor cells developvarious escape mechanisms to evade the T-cell-mediated immunotherapy. Inone such escape mechanism, a cancer cell population which originallyexpressed a normal level of HLA on the cell surface reduces oreliminates the expression of HLA class I and/or HLA class II within thecancer cell population. The absence of HLA or the reduction of HLAexpression impairs the recognition of cancer cells by T-cells and thusreduces subsequent cell lysis mediated by T-cells.

NK cells are at the core of innate immunity and can respond rapidly toviral infection or tumor formation by mediating the lysis of tumor cellsand virally infected cells via natural cytotoxicity andantibody-dependent cellular cytotoxicity (“ADCC”). While a typicalimmune cell requires recognition of the major histocompatibility complex(“MHC”) or HLA on the cell surface before triggering cytolyticresponses, NK cells may recognize the infected cells or tumor in theabsence of HLA on the cell surface. This unique feature of NK cellsrenders NK cells suitable for targeting those cancer cells with no orreduced HLA expression.

In one embodiment, a patient having a plurality of HLA-negative cancercells or cancer cells with reduced HLA expression is treated withIFN-alpha in an amount sufficient to expand and/or activate immune cellssuch that the activated and/or expanded immune cells kill one or more ofHLA-negative cancer cells or the cancer cells with reduced HLAexpression. In another embodiment, a sufficient amount, or asub-therapeutic amount of IFN-alpha can expand and/or activate theendogenous immune cells, including, but not limited to, NK cells in ahost.

In another embodiment, NK cells can be expanded or activated ex vivo byIFN-alpha before they are administered back to the host to kill theHLA-negative cancer cells or the cancer cells with reduced expression ofHLA compared to a normal control. NK cells can be administered to anindividual by absolute numbers of cells; e.g., said individual can beadministered from about 1000 cells/injection to up to about 10 billioncells/injection, such as at about, at least about, or at most about,1×10⁸, 1×10⁷, 5×10⁷, 1×10⁶, 5×10⁶, 1×10⁵, 5×10⁵, 1×10 ⁴, 5×10⁴, 1×10³,5×10³ (and so forth) NK cells per injection, or any ranges between anytwo of the numbers, end points inclusive. In other embodiments, theamount of NK cells injected per dose may be calculated by m² of bodysurface area, including 1×10¹¹, 1×10¹⁰, 1×10⁹, 1×10⁸, 1×10⁷ (and soforth) NK cells per m². The average person is 1.6-1.8 m². In otherembodiments, said individual can be administered from about 1000cells/injection/m² to up to about 10 billion cells/injection/m², such asat about, at least about, or at most about, 1×10⁸/m², 1×10⁷/m²,5×10⁷/m², 1×10⁶ m², 5×10⁶/m², 1×10⁵/m², 5×10⁵/m², 1×10⁴/m², 5×10⁴/m²,1×10³/m², 5×10³/m² (and so forth) NK cells per injection, or any rangesbetween any two of the numbers, end points inclusive. In otherembodiments, NK-92 cells can be administered to such an individual byrelative numbers of cells; e.g., said individual can be administeredabout 1000 cells to up to about 10 billion cells per kilogram of theindividual, such as at about, at least about, or at most about, 1×10⁸,1×10⁷, 5×10⁷, 1×10⁶, 5×10⁶, 1×10⁵, 5×10⁵, 1×10⁴, 5×10⁴, 1×10³, 5×10³(and so forth) NK cells per kilogram of the individual, or any rangesbetween any two of the numbers, end points inclusive.

The immune cells of the present disclosure can be isolated from anysource. In some embodiments, the source of the immune cells isautologous, allogeneic, or xenographic, or combinations thereof. Theimmune cells may be prepared ex vivo by extracting or otherwiseisolating autologous immune cells from blood, bone marrow, or otherimmune cell-containing organs of a patient having a cancerous tumor orother cancer, according to methods known in the art. For example, suchmethods include, but are not intended to be limited to, apheresistechniques, specifically leukapheresis. In another embodiment of theinvention, the NK cells can be autologous or allogeneic NK cells.“Autologous” NK cells are cells derived from the patient. “Allogeneic”NK cells are derived from another individual, having non-identical genesat one or more loci. If the NK cells are derived from an identical twin,they may be termed “syngeneic.”

Additionally, commercially available kits may be utilized for theextraction of NK cells, such as with EasySep™ Human NK Cell IsolationKit available from STEMCELL™ Technologies, Inc., British Columbia,CANADA.

Natural Killer (NK) Cells

Natural killer (NK) cells are a class of lymphocytes that typicallycomprise approximately 10% of the lymphocytes in a human. NK cellsprovide an innate cellular immune response against tumor and infected(target) cells. NK cells, which are characterized as having a CD3−/CD56+phenotype, display a variety of activating and inhibitory cell surfacereceptors. NK cell inhibitory receptors predominantly engage with majorhistocompatibility complex class I (“MHC-I”) proteins on the surface ofa normal cell to prevent NK cell activation. The MHC-I molecules definecells as “belonging” to a particular individual. It is thought that NKcells can be activated only by cells on which these “self” MHC-Imolecules are missing or defective, such as is often the case for tumoror virus-infected cells.

NK cells are triggered to exert a cytotoxic effect directly against atarget cell upon binding or ligation of an activating NK cell receptorto the corresponding ligand on the target cell. The cytotoxic effect ismediated by secretion of a variety of cytokines by the NK cells, whichin turn stimulate and recruit other immune system agents to act againstthe target. Activated NK cells also lyse target cells via the secretionof the enzymes perforin and granzyme, stimulation ofapoptosis-initiating receptors, and other mechanisms.

NK cells have been evaluated as an immunotherapeutic agent in thetreatment of certain cancers. NK cells used for this purpose may beautologous or non-autologous (i.e., from a donor).

In one embodiment, the NK cells used in the compositions and methodsherein are autologous NK cells. In one embodiment, the NK cells used inthe compositions and methods herein are non-autologous NK cells.

In one embodiment, the NK cells used in the compositions and methodsherein are genetically modified NK cells. NK cells can be geneticallymodified by insertion of genes or RNA into the cells such that the cellsexpress one or more proteins that are not expressed by wild type NKcells. In one embodiment, the NK cells are genetically modified toexpress a chimeric antigen receptor (CAR). In a preferred embodiment,the CAR is specific for the cancer being targeted by the method orcomposition.

Non-limiting examples of modified NK cells can be found, for example, inGlienke, et al. 2015, Advantages and applications of CAR-expressingnatural killer cells, Frontiers in Pharmacol. 6, article 21; PCT PatentPub. Nos. WO 2013154760 and WO 2014055668; each of which is incorporatedherein by reference in its entirety.

In some embodiments, the NK cells are an NK cell line. NK cell linesinclude, without limitation, NK-92, NK-YS, KHYG-1, NKL, NKG, SNK-6, andIMC-1. See, Klingemann et al. Front Immunol. 2016; 7: 91, which isincorporated herein by reference in its entirety.

NK-92 Cells

The NK-92 cell line was discovered in the blood of a subject sufferingfrom a non-Hodgkins lymphoma. NK-92 cells lack the major inhibitoryreceptors that are displayed by normal NK cells, but retain a majorityof the activating receptors. NK-92 cells are cytotoxic to asignificantly broader spectrum of tumor and infected cell types than areNK cells and often exhibit higher levels of cytotoxicity toward thesetargets. NK-92 cells do not, however, attack normal cells, nor do theyelicit an immune rejection response. In addition, NK-92 cells can bereadily and stably grown and maintained in continuous cell culture and,thus, can be prepared in large quantities under c-GMP compliant qualitycontrol. This combination of characteristics has resulted in NK-92 beingentered into presently on-going clinical trials for the treatment ofmultiple types of cancers.

NK-92 cells used in the compositions and methods described herein may bewild type (i.e., not genetically modified) NK-92 cells or geneticallymodified NK-92 cells. NK-92 cells can be genetically modified byinsertion of genes or RNA into the cells such that the cells express oneor more proteins that are not expressed by wild type NK-92 cells. In oneembodiment, NK-92 cells are genetically modified to express a chimericantigen receptor (CAR) on the cell surface. In a preferred embodiment,the CAR is specific for the cancer being targeted by the method orcomposition. In one embodiment, NK-92 cells are genetically modified toexpress an Fe receptor on the cell surface. In a preferred embodiment,the NK-92 cell expressing the Fe receptor can mediate antibody-dependentcell-mediated cytotoxicity (ADCC). In one embodiment, the Fe receptor isCD16. In one embodiment, NK-92 cells are genetically modified to expressa cytokine (e.g., IL-2).

In one embodiment, the modified NK-92 cell is administered incombination with an antibody specific for the cancer to be treated. In apreferred embodiment, the modified NK-92 cell administered incombination with the antibody is competent to mediate ADCC.

Examples of NK-92 cells are available from the American Type CultureCollection (ATCC) as ATCC CRL-2407.

Non-limiting examples of modified NK-92 cells are described, forexample, in U.S. Pat. Nos. 7,618,817 and 8,034,332; and U.S. Patent Pub.Nos. 2002/0068044 and 2008/0247990, each of which is incorporated hereinby reference in its entirety. Examples of modified NK-92 cells areavailable from ATCC as ATCC CRL-2408, ATCC CRL-2409, PTA-6670, PTA-6967,PTA-8837, and PTA-8836. Non-limiting examples of CAR-modified NK-92cells can be found, for example, in Glienke, et al. 2015, Advantages andapplications of CAR-expressing natural killer cells, Frontiers inPharmacol. 6, article 21; which is incorporated herein by reference inits entirety.

Interferon

Interferons (“IFNs”) have long been recognized for their roles inregulating the immune response to infection inflammation and tumorformation. Human interferons are grouped into three classes based ontheir cellular origin and antigenicity: a-interferon (leukocytes),(3-interferon (fibroblasts) and γ-interferon (B-cells). Recombinantforms of each group have been developed and are commercially available.Subtypes in each group are based on antigenic/structuralcharacteristics. There are at least 24 IFN alpha subtypes identified sofar, including IFN-alpha 1, IFN-alpha 2, IFN-alpha 4, IFN-alpha 5,IFN-alpha 6, IFN-alpha 7, IFN-alpha 8, IFN-alpha 10, IFN-alpha 14,IFN-alpha 16, IFN-alpha 17, or IFN-alpha 21. Among them, IFN-α1, IFN-α2,IFN-α8, IFN-α10, IFN-α14 and IFN-α21 are the major subtypes ofIFN-alphas. In one embodiment, the IFN-alpha is IFN-alpha 1, IFN-alpha2, IFN-alpha 4, IFN-alpha 5, IFN-alpha 6, IFN-alpha 7, IFN-alpha 8,IFN-alpha 10,

IFN-alpha 14, IFN-alpha 16, IFN-alpha 17, or IFN-alpha 21. In anotherembodiment, IFN-alpha is IFN-alpha 2, IFN-alpha 8, or IFN-alpha 10. Inother preferred embodiments, the IFN-alpha is IFN-alpha 8.

IFNs, e.g., IFN-alphas, can enhance immune cell cytotoxicity, migration,and cytokine production and inhibit tumor growth. The conditions thatcan be treated with IFNs include, but are not limited to, cellproliferation disorders, in particular cancer (e.g., hairy cellleukemia, Kaposi's sarcoma, chronic myelogenous leukemia, multiplemyeloma, basal cell carcinoma and malignant melanoma, ovarian cancer,cutaneous T-cell lymphoma), and viral infections. Viral infections whichmay be treated in accordance with the invention include hepatitis A,hepatitis B, hepatitis C, other non-A/non-B hepatitis, herpes virus,

Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes simplex, humanherpes virus type 6 (HHV-6)), papilloma, poxvirus, picornavirus,adenovirus, rhinovirus, human T lymphotropic virus-type 1 and 2(HTLV-1/-2), human rotavirus, rabies, retroviruses including humanimmunodeficiency virus (HIV), encephalitis and respiratory viralinfections.

A number of immune cells can be stimulated, activated, and expanded byIFNs. For example, IFNs can enhance the cytotoxic activities of NKlymphocytes, which is critical for the primary innate immune systemagainst viral and bacterial infections and tumorigenesis. Markovic SN etal (1991) Cancer Research, 51:1124. Moreover, the tumor cells from theIFN receptor-deficient animal models are unresponsive to the NK-mediatedcell lysis. Swann JB, et al (2007) J of Immun, 178 (12):7540-7549.Therefore, the IFNs, including IFN-alpha, may be critical forcontrolling NK cell-mediated antitumor responses.

In one embodiment, a sufficient amount, or a sub-therapeutic amount ofIFN-alpha can expand and/or activate the endogenous immune cells,including but not limited to NK cells in a host. The amount of IFN-alphainjected per dose may be calculated by square meter (m²) of body surfacearea, including 1×10⁷, 1×10⁶, 1×10⁵, 1×10⁴, 1×10³, 1×10² (and so forth)units per m² (U/m²). The average person is 1.6-1.8 m².

In other embodiments, said individual can be administered from about 100units/injection/m² to up to about 10 million units/injection/m², such asat about, at least about, or at most about, 1×10⁷ U/m², 1×10⁶ U/m²,1×10⁵ U/m², 1×10⁴ U/m², 1×10³ U/m², 1×10² U/m² (and so forth) IFN-alphaper injection, or any ranges between any two of the numbers, end pointsinclusive. In other embodiments, said individual can be administered atabout, at least about, or at most about, 5×10⁶ U/m², 5×10⁵ U/m², 5×10⁴U/m² 5×10³ U/m², 5×10² U/m² (and so forth) IFN-alpha per injection, orany ranges between any two of the numbers, end points inclusive.

In one embodiment, the sub-therapeutic amount of the IFN-alpha is about1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%,about 35%, about 40%, about 45%, about 50%, about 55%, or about 60% of atherapeutic dosage of the IFN-alpha, or any ranges between any two ofthe numbers, end points inclusive. In another embodiment, the IFN-alphais provided in an amount ranging from about 5×10¹ U/m² to about 2×10⁵U/m².

The amount of IFN-alpha used to expand and/or activate immune cells exvivo can be calculated by the number of cells. The concentration ofIFN-alpha for the ex vivo incubation of immune cells, e.g. NK cells, canbe about 1×10¹ U/1×10⁶ cells to 1×10⁶ U/1×10⁶ cell, such as at about, atleast about, or at most about, 1×10⁸ U/1×10⁶ cells, 1×10⁷ U/1×10⁶ cells,5×10⁷ U/1×10⁶ cells, 1×10⁶ U/1×10⁶ cells, 5×10⁶ U/1×10⁶ cells, 1×10⁵U/1×10⁶ cells, 5×10⁵ U/1×10⁶ cells, 1×10⁴ U/1×10⁶ cells, 5×10⁴ U/1×10⁶cells, 1×10³ U/1×10⁶ cells, 5×10³ U/1×10⁶ cells, 1×10² U/1×10⁶ cells,5×10² U/1×10⁶ cells, 1×10¹ U/1×10⁶ cells, 5×10¹ U/1×10⁶ cells (and soforth), or any ranges between any two of the numbers, end pointsinclusive. In one embodiment, the NK cells are incubated ex vivo withthe IFN-alpha at a concentration from 5×10¹ U/1×10⁶ cells to 1×10⁴U/1×10⁶ cells.

Homing Receptor

In some embodiments, the immune cells are modified to express a tumorcell homing receptor on the outer cell surface of the immune cell. Thehoming receptor may be, for example, a chimeric antigen receptor, an Fereceptor, or combinations thereof. In some embodiments the CAR targets acancer-associated antigen. In other embodiments, at least a portion ofthe immune cells express an endogenous tumor cell homing receptor thatis not CXCR4.

In one aspect of the disclosure, the immune cell is modified to expressa chimeric antigen receptor (CAR). In some embodiments, the immune cellis transformed with a nucleic acid encoding a CAR, wherein the CAR isexpressed on the outer cell surface of the immune cell. In someembodiments, the immune cell is a T-cell, for example, an activatedT-cell.

Any CAR known to one of skill in the art now or in the future isencompassed by the present disclosure. In one embodiment, the CAR isspecific for a tumor-specific antigen. Tumor-specific antigens can alsobe referred to as cancer-specific antigen. In one embodiment, the CAR isspecific for a tumor-associated antigen. Tumor-associated antigens canalso be referred to as cancer-associated antigen. A tumor-specificantigen is a protein or other molecule that is unique to cancer cells,while a tumor-associated antigen is an antigen that is highly correlatedwith certain tumor cells and typically are found at higher levels on atumor cell as compared to on a normal cell. Tumor-specific antigens aredescribed, by way of non-limiting example, in U.S. Patent No. 8,399,645;U.S. Pat. No. 7,098,008; WO 1999/024566; WO 2000/020460; and WO2011/163401, each of which is incorporated herein by reference in itsentirety. In addition, examples of some known CARs are disclosure inTable 2. In one embodiment, the CAR targets a tumor-associated antigenselected from the group consisting of a-folate receptor, CAIX, CD19,CD20, CD30, CD33, CEA, EGP-2, erb-B2, erb-B 2,3,4, FBP, GD2, GD3,Her2/neu, IL-13R-a2, k-light chain, LeY, MAGE-A1, Mesothelin, and PSMA.

In some embodiments, the CAR recognizes an antigen associated with aspecific cancer type selected from the group consisting of ovariancancer, renal cell carcinoma, B-cell malignancies, Acute lymphoblasticleukemia (ALL), chronic lymphocytic leukemia (CLL), B-cell malignancies,refractory follicular lymphoma, mantle cell lymphoma, indolent B-celllymphoma, acute myeloid leukemia (AML), Hodgkin lymphoma, cervicalcarcinoma, breast cancer, colorectal cancer, prostate cancer,neuroblastoma, melanoma, rhabdomyosarcoma, medulloblastoma,adenocarcinomas, and tumor neovasculature.

TABLE 2 Chimeric Antigen Receptors CARs Target antigen Associatedmalignancy Receptor type generation α-Folate receptor Ovarian cancerScFv-FcεRIγCAIX First CAIX Renal cell carcinoma ScFv-FcεRIγ First CAIXRenal cell carcinoma ScFv-FcεRIγ Second CD19 B-cell malignanciesScFv-CD3ζ (EBV) First CD19 B-cell malignancies, CLL ScFv-CD3ζ First CD19B-ALL ScFv-CD28-CD3ζ Second CD19 ALL CD3ζ (EBV) First CD19 ALL post-HSCTScFv-CD28-CD3ζ Second CD19 Leukemia, lymphoma, CLL ScFv-CD28-CD3ζ vs.First and CD3ζ Second CD19 B-cell malignancies ScFv-CD28-CD3ζ SecondCD19 B-cell malignancies post- ScFv-CD28-CD3ζ Second HSCT CD19Refractory Follicular ScFv-CD3ζ First Lymphoma CD19 B-NHL ScFv-CD3ζFirst CD19 B-lineage lymphoid ScFv-CD28-CD3ζ Second malignanciespost-UCBT CD19 CLL, B-NHL ScFv-CD28-CD3ζ Second CD19 B-cellmalignancies, ScFv-CD28-CD3ζ Second CLL, B-NHL CD19 ALL, lymphomaScFv-41BB-CD3ζ vs First and CD3ζ second CD19 ALL ScFv-41BB-CD3ζ SecondCD19 B-cell malignancies ScFv-CD3ζ (Influenza First MP-1) CD19 B-cellmalignancies ScFv-CD3ζ (VZV) First CD20 Lymphomas ScFv-CD28-CD3ζ SecondCD20 B-cell malignancies ScFv-CD4-CD3ζ Second CD20 B-cell lymphomasScFv-CD3ζ First CD20 Mantle cell lymphoma ScFv-CD3ζ First CD20 Mantlecell lymphoma, CD3ζ/CD137/CD28 Third indolent B-NHL CD20 indolent B celllymphomas ScFv-CD28-CD3ζ Second CD20 Indolent B cell lymphomasScFv-CD28-41BB- Third CD3ζ CD22 B-cell malignancies ScFV-CD4-CD3ζ SecondCD30 Lymphomas ScFv-FcεRIγ First CD30 Hodgkin lymphoma ScFv-CD3ζ (EBV)First CD33 AML ScFv-CD28-CD3ζ Second CD33 AML ScFv-41BB-CD3ζ SecondCD44v7/8 Cervical carcinoma ScFv-CD8-CD3ζ Second CEA Breast cancerScFv-CD28-CD3ζ Second CEA Colorectal cancer ScFv-CD3ζ First CEAColorectal cancer ScFv-FceRIγ First CEA Colorectal cancer ScFv-CD3ζFirst CEA Colorectal cancer ScFv-CD28-CD3ζ Second CEA Colorectal cancerScFv-CD28-CD3ζ Second EGP-2 Multiple malignancies scFv-CD3ζ First EGP-2Multiple malignancies scFv-FcεRIγ First EGP-40 Colorectal cancerscFv-FcεRIγ First erb-B2 Colorectal cancer CD28/4-1BB-CD3ζ Third erb-B2Breast and others ScFv-CD28-CD3ζ Second erb-B2 Breast and othersScFv-CD28-CD3ζ Second (Influenza) erb-B2 Breast and othersScFv-CD28mut-CD3ζ Second erb-B2 Prostate cancer ScFv-FcεRIγ First erb-B2,3,4 Breast and others Heregulin-CD3ζ Second erb-B 2,3,4 Breast andothers ScFv-CD3ζ First FBP Ovarian cancer ScFv-FcεRIγ First FBP Ovariancancer ScFv-FcεRIγ First (alloantigen) Fetal Rhabdomyosarcoma ScFv-CD3ζFirst acetylcholine receptor GD2 Neuroblastoma ScFv-CD28 First GD2Neuroblastoma ScFv-CD3ζ First GD2 Neuroblastoma ScFv-CD3ζ First GD2Neuroblastoma ScFv-CD28-OX40- Third CD3ζ GD2 Neuroblastoma ScFv-CD3ζ(VZV) First GD3 Melanoma ScFv-CD3ζ First GD3 Melanoma ScFv-CD3ζ FirstHer2/neu Medulloblastoma ScFv-CD3ζ First Her2/neu Lung malignancyScFv-CD28-CD3ζ Second Her2/neu Advanced osteosarcoma ScFv-CD28-CD3ζSecond Her2/neu Glioblastoma ScFv-CD28-CD3ζ Second IL-13R-a2 GliomaIL-13-CD28-4-1BB- Third CD3ζ IL-13R-a2 Glioblastoma IL-13-CD3ζ SecondIL-13R-a2 Medulloblastoma IL-13-CD3ζ Second KDR Tumor neovasculatureScFv-FcεRIγ First k-light chain B-cell malignancies ScFv-CD3ζ Firstk-light chain (B-NHL, CLL) ScFv-CD28-CD3ζ vs Second CD3ζ LeY CarcinomasScFv-FcεRIγ First LeY Epithelial derived tumors ScFv-CD28-CD3ζ Second L1cell adhesion Neuroblastoma ScFv-CD3ζ First molecule MAGE-A1 MelanomaScFV-CD4-FcεRIγ Second MAGE-A1 Melanoma ScFV-CD28-FcεRIγ SecondMesothelin Various tumors ScFv-CD28-CD3ζ Second Mesothelin Varioustumors ScFv-41BB-CD3ζ Second Mesothelin Various tumors ScFv-CD28-41BB-Third CD3ζ Murine CMV Murine CMV Ly49H-CD3ζ Second infected cells MUC1Breast, Ovary ScFV-CD28-OX40- Third CD3ζ NKG2D ligands Various tumorsNKG2D-CD3ζ First Oncofetal antigen Various tumors ScFv-CD3ζ First (h5T4)(vaccination) PSCA Prostate carcinoma ScFv-b2c-CD3ζ Second PSMAProstate/tumor vasculature ScFv-CD3ζ First PSMA Prostate/tumorvasculature ScFv-CD28-CD3ζ Second PSMA Prostate/tumor vasculatureScFv-CD3ζ First TAA targeted by Various tumors FceRI-CD28-CD3ζ Third mAhIgE (+a-TAA IgE mAb) TAG-72 Adenocarcinomas scFv-CD3ζ First VEGF-R2Tumor neovasculature scFv-CD3ζ First

Chemotherapy Agents

In one aspect of the present invention, the modified immune cells or IFNare administered in combination with a chemotherapy agent. Thechemotherapy agent may be any agent having a therapeutic effect on oneor more types of cancer. Many chemotherapy agents are currently known inthe art. Types of chemotherapy drugs include, by way of non-limitingexample, alkylating agents, antimetabolites, anti-tumor antibiotics,topoisomerase inhibitors, mitotic inhibitors, corticosteroids, and thelike.

Non-limiting examples of chemotherapy drugs include: nitrogen mustards,such as mechlorethamine (nitrogen mustard), chlorambucil,cyclophosphamide (Cytoxan®), ifosfamide, and melphalan); Nitrosoureas,such as streptozocin, carmustine (BCNU), and lomustine; alkylsulfonates, such as busulfan; Triazines, such as dacarbazine (DTIC) andtemozolomide (Temodar®); ethylenimines, such as thiotepa and altretamine(hexamethylmelamine); platinum drugs, such as cisplatin, carboplatin,and oxalaplatin; 5-fluorouracil (5-FU); 6-mercaptopurine (6-MP);Capecitabine (Xeloda®); Cytarabine (Ara-C®); Floxuridine; Fludarabine;Gemcitabine (Gemzar®); Hydroxyurea; Methotrexate; Pemetrexed (Alimta®);anthracyclines, such as Daunorubicin, Doxorubicin (Adriamycin®),Epirubicin, Idarubicin; Actinomycin-D; Bleomycin; Mitomycin-C;Mitoxantrone; Topotecan; Irinotecan (CPT-11); Etoposide (VP-16);Teniposide; Mitoxantrone; Taxanes: paclitaxel (Taxol®) and docetaxel(Taxotere®); Epothilones: ixabepilone (Ixempra®); Vinca alkaloids:vinblastine (Velban®), vincristine (Oncovin®), and vinorelbine(Navelbine®); Estramustine (Emcyt®); Prednisone; Methylprednisolone(Solumedrol®); Dexamethasone (Decadron®); L-asparaginase; bortezomib(Velcade®). Additional chemotherapy agents are listed, for example, inU.S. Patent Application Pub. No. 2008/0300165, which is incorporatedherein by reference in its entirety.

Doses and administration protocols for chemotherapy drugs are well-knownin the art. The skilled clinician can readily determine the properdosing regimen to be used, based on factors including the chemotherapyagent(s) administered, type of cancer being treated, stage of thecancer, age and condition of the patient, patient size, location of thetumor, and the like.

Radiotherapy Agents

In one aspect of the present invention, the modified immune cells or IFNare administered in combination with a radiotherapeutic agent. Theradiotherapeutic agent may be any such agent having a therapeutic effecton one or more types of cancer. Many radiotherapeutic agents arecurrently known in the art. Types of radiotherapeutic drugs include, byway of non-limiting example, X-rays, gamma rays, and charged particles.In one embodiment, the radiotherapeutic agent is delivered by a machineoutside of the body (external-beam radiation therapy). In a preferredembodiment, the radiotherapeutic agent is placed in the body near thetumor/cancer cells (brachytherapy) or is a systemic radiation therapy.

External-beam radiation therapy may be administered by any means.Exemplary, non-limiting types of external-beam radiation therapy includelinear accelerator-administered radiation therapy, 3-dimensionalconformal radiation therapy (3D-CRT), intensity-modulated radiationtherapy (IMRT), image-guided radiation therapy (IGRT), tomotherapy,stereotactic radiosurgery, photon therapy, stereotactic body radiationtherapy, proton beam therapy, and electron beam therapy.

Internal radiation therapy (brachytherapy) may be by any technique oragent. Exemplary, non-limiting types of internal radiation therapyinclude any radioactive agents that can be placed proximal to or withinthe tumor, such as Radium-226 (Ra-226), Cobalt-60 (Co-60), Cesium-137(Cs-137), cesium-131, Iridium-192 (Ir-192), Gold-198 (Au-198),Iodine-125 (I-125), palladium-103, yttrium-90, etc. Such agents may beadministered by seeds, needles, or any other route of administration,and may be temporary or permanent. Systemic radiation therapy may be byany technique or agent. Exemplary, non-limiting types of systemicradiation therapy include radioactive iodine, ibritumomab tiuxetan(Zevalin®), tositumomab and iodine I 131 tositumomab (Bexxar®),samarium-153-lexidronam (Quadramet®), strontium-89 chloride(Metastron®), metaiodobenzylguanidine, lutetium-I 77, yttrium-90,strontium-89, and the like.

In one embodiment, a radiosensitizing agent is also administered to thepatient. Radiosensitizing agents increase the damaging effect ofradiation on cancer cells.

Doses and administration protocols for radiotherapy agents arewell-known in the art. The skilled clinician can readily determine theproper dosing regimen to be used, based on factors including theagent(s) administered, type of cancer being treated, stage of thecancer, location of the tumor, age and condition of the patient, patientsize, and the like.

Anti-Cancer Vaccines

In one aspect of the present invention, the modified immune cells or IFNare administered in combination with an anti-cancer vaccine (also calledcancer vaccine). Anti-cancer vaccines are vaccines that either treatexisting cancer or prevent development of a cancer by stimulating animmune reaction to kill the cancer cells. In a preferred embodiment, theanti-cancer vaccine treats existing cancer.

The anti-cancer vaccine may be any such vaccine having a therapeuticeffect on one or more types of cancer. Many anti-cancer vaccines arecurrently known in the art. Such vaccines include, without limitation,dasiprotimut-T, Sipuleucel-T, talimogene laherparepvec, HSPPC-96 complex(Vitespen), L-BLP25, gp100 melanoma vaccine, and any other vaccine thatstimulates an immune response to cancer cells when administered to apatient.

Antibodies

In one aspect of the present invention, the modified immune cells or IFNare administered in combination with an anti-tumor antibody. That is,antibodies specific for a particular type of cancer (e.g., a cellsurface protein expressed by the target cancer cells) can beadministered to a patient having cancer. The antibodies may bemonoclonal antibodies, polyclonal antibodies, chimeric antibodies,antibody fragments, human antibodies, humanized antibodies, or non-humanantibodies (e.g. murine, goat, primate, etc.). The therapeutic antibodymay be specific for any tumor-specific or tumor-associated antigen. See,e.g. Scott et al., Cancer Immunity 2012, 12:14, which is incorporatedherein by reference in its entirety.

Non-limiting examples include trastuzumab (Herceptin®), bevacizumab(Avastin®), cetuximab (Erbitux®), panitumumab (Vectibix®), ipilimumab(Yervoy®), rituximab (Rituxan®), alemtuzumab (Campath®), ofatumumab(Arzerra®), gemtuzumab ozogamicin (Mylotarg®), brentuximab vedotin(Adcetris®),⁹⁰Y-ibritumomab tiuxetan (Zevalin®), and ¹³¹1-tositumomab(Bexxar®).

Additional antibodies are provided in Table 1.

TABLE 1 Anti-cancer antibodies Proprietary Indication first name Tradename Target; Format approved or reviewed Necitumumab (Pending) EGFR;Human IgG1 Non-small cell lung cancer Nivolumab Opdivo PD1; Human IgG4Melanoma Dinutuximab (Pending) GD2; Chimeric IgG1 NeuroblastomaBlinatumomab Blincyto CD19, CD3; Murine Acute lymphoblastic bispecifictandem scFv leukemia Pembrolizumab Keytruda PD1; Humanized IgG4 MelanomaRamucirumab Cyramza VEGFR2; Human IgG1 Gastric cancer ObinutuzumabGazyva CD20; Humanized IgG1; Chronic lymphocytic Glycoengineeredleukemia Ado-trastuzumab Kadcyla HER2; humanized IgG1; Breast canceremtansine immunoconjugate Pertuzumab Perjeta HER2; humanized IgG1 BreastCancer Brentuximab Adcetris CD30; Chimeric IgG1; Hodgkin lymphoma,vedotin immunoconjugate systemic anaplastic large cell lymphomaIpilimumab Yervoy CTLA-4; Human IgG1 Metastatic melanoma OfatumumabArzerra CD20; Human IgG1 Chronic lymphocytic leukemia

Immune Checkpoint Inhibitors

In one aspect of the present invention, the modified immune cells or IFNare administered in combination with a checkpoint inhibitor. Immunecheckpoint proteins are made by some types of immune system cells, suchas T cells, and some cancer cells. These proteins, which can prevent Tcells from killing cancer cells, are targeted by checkpoint inhibitors.Checkpoint inhibitors increase the T cells' ability to kill the cancercells. Examples of checkpoint proteins found on T cells or cancer cellsinclude PD-1/PD-L1 and CTLA-4/B 7-1/B7-2.

In one embodiment, the checkpoint inhibitor is an antibody to acheckpoint protein, e.g., PD-1, PDL-1, or CTLA-4. Checkpoint inhibitorantibodies include, without limitation, BMS-936559, MPDL3280A,MedI-4736, Lambrolizumab, Alemtuzumab, Atezolizumab, Ipilimumab,Nivolumab, Ofatumumab, Pembrolizumab, and Rituximab.

Treatment Methods

In one aspect is provided a method for treating a patient having aplurality of HLA-negative cancer cells or cancer cells with reduced HLAexpression with interferon-alpha (“IFN-alpha”) in an amount sufficientto expand and/or activate immune cells such that the activated and/orexpanded immune cells kill one or more of HLA-negative cancer cells orthe cancer cells with reduced HLA expression.

In one embodiment, the method comprises identifying a patient having acancer with reduced HLA expression. In one embodiment, the methodcomprises identifying a patient having a cancer which does not expressHLA. In one embodiment, the method comprises determining HLA expressionof a cancer in a patient before and/or after treatment of the patientwith an anti-cancer agent, such as a T-cell therapy. In one embodiment,the method comprises identifying a patient having a cancer amenable totreatment with IFN-alpha as described herein by determining HLAexpression of a cancer in a patient before and/or after treatment. Inone embodiment, the method comprises selecting a patient having a cancerthat does not express HLA or that has reduced HLA expression.

In one embodiment, the amount sufficient to expand and/or activateimmune cells is a sub-therapeutic amount of the IFN-alpha. It iscontemplated that administration of a sub-therapeutic amount ofIFN-alpha can still activate, or expand the immune cells, including NKcells, such that the activated and/or expanded immune cells can kill oneor more of HLA-negative cancer cells or cancer cells with reduced HLAexpression. In one embodiment, the sub-therapeutic amount of IFN-alphacan be about 1%, about 5%, about 10%, about 15%, about 20%, about 25%,about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, orabout 60% of a therapeutic dosage of the IFN-alpha. In anotherembodiment, the IFN-alpha is administered in an amount ranging fromabout 5×10¹ U/m² to about 2×10⁵ U/m². Administration of asub-therapeutic amount of IFN-alpha allows for greater targeting of theimmune cells (e.g., NK cells) to the tumor, or decreasing any potentialside effects associated with IFN-alpha, or both. In one embodiment, toexpand, activate, and/or stimulate the immune cells, the IFN-alpha canbe administered orally, via vein injection, muscle injection, peritonealinjection, subcutaneous injection, nasal or mucosal administration, orby inhalation.

As discussed, cancers may develop one or more escape mechanisms to evadeimmunotherapy, including T-cell-mediated therapy, by reducing theexpression of HLA on the cancer cell surface. It is contemplated thatthe methods described herein result in recognition and killing of thecancer cells with no or reduced HLA expression. In one aspect of theinvention, the cancer comprise a population of cancer cells thatinitially express an HLA prior to comprising a population that isHLA-negative or have reduced HLA expression. In one embodiment, thepatient has received or is selected based upon having received animmunotherapy for the cancer. In one embodiment, the immunotherapy is aT-cell therapy. In one embodiment, the immunotherapy targeted the HLApositive cells in the patient's cancer cell population, resulting in areduction in HLA positive cells within the population.

In one embodiment, the IFN-alpha is administered as part of acombination therapy. In another embodiment, the combination therapycomprises one or more of chemotherapy, radiotherapy, and immunotherapy.

In addition to expanding and activating the endogenous immune cells,e.g., NK cells, IFN-alpha can also expand and activate the immune cellsex vivo before the expanded and activated immune cells are administeredback to a patient with cancers comprising cells with no or reducedexpression of HLA. One aspect of the invention relates to a method fortreating a cancer, which comprises administering an immune cell to asubject in need thereof, wherein said immune cell is activated byIFN-alpha, further wherein the cancer has no or reduced expression of aHLA compared to a normal control. In one embodiment, the immune cell isa T-cell, a B-cell, or a NK-cell. In one embodiment, the immune cells,including the NK cells, are activated by incubating the immune cell withthe IFN-alpha ex vivo. In another embodiment, the immune cell isincubated ex vivo with the IFN-alpha at a concentration from5×10¹U/1×10⁶ cells to 1×10⁴U/1×10⁶ cells.

In one embodiment, the HLA is a class I HLA antigen, a class II HLAantigen, or a class III HLA antigen. Preferably, the HLA is a class IHLA antigen and class II HLA antigen. In another embodiment, the sourceof the NK cells or the immune cells is autologous, allogeneic, orxenographic. In another embodiment, the NK cells or the immune cells areadministered intravenously, intraperitoneally, intramuscularly, andsubcutaneously.

In one embodiment, IFN-alpha can be one or more of IFN-alpha 1,IFN-alpha 2, IFN-alpha 4, IFN-alpha 5, IFN-alpha 6, IFN-alpha 7,IFN-alpha 8, IFN-alpha 10, IFN-alpha 14, IFN-alpha 16, IFN-alpha 17, orIFN-alpha 21. Preferably, IFN-alpha is IFN-alpha 2, IFN-alpha 8, orIFN-alpha 10. In other preferred embodiments, the IFN-alpha is IFN-alpha8.

In one embodiment, the IFN is a synthetic or recombinant IFN-alpha.

In one embodiment, the immune cells, such as NK cells, are administeredin combination with an anti-tumor agent. In another embodiment, theanti-tumor agent is a chemotherapy agent, a radiotherapy agent, ananti-cancer antibody, an immune checkpoint inhibitor, or an anti-cancervaccine.

In one embodiment, the immune cell is modified to express a tumor cellhoming receptor on the outer cell surface of the immune cell. In anotherembodiment, the tumor cell homing receptor is a chimeric antigenreceptor, an Fe receptor, or combinations thereof. In one embodiment,the immune cell is a T-cell, a B-cell, or a NK cell. In anotherembodiment, the patient or subject is human.

Cancers or tumors that can be treated by the cells and methods describedherein include, but are not limited to: biliary tract cancer; braincancer, including glioblastomas and medulloblastomas; breast cancer;cervical cancer; choriocarcinoma; colon cancer; endometrial cancer;esophageal cancer; gastric cancer; hematological neoplasms, includingacute lymphocytic and myelogenous leukemia; multiple myeloma; AIDSassociated leukemias and adult T-cell leukemia lymphoma; intraepithelialneoplasms, including Bowen's disease and Paget's disease; liver cancer(hepatocarcinoma); lung cancer; lymphomas, including Hodgkin's diseaseand lymphocytic lymphomas; neuroblastomas; oral cancer, includingsquamous cell carcinoma; ovarian cancer, including those arising fromepithelial cells, stromal cells, germ cells and mesenchymal cells;pancreas cancer; prostate cancer; rectal cancer; sarcomas, includingleiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma andosteosarcoma; skin cancer, including melanoma, Kaposi's sarcoma,basocellular cancer and squamous cell cancer; testicular cancer,including germinal tumors (seminoma, non-seminoma[teratomas,choriocarcinomas]), stromal tumors and germ cell tumors; thyroid cancer,including thyroid adenocarcinoma and medullar carcinoma; and renalcancer including adenocarcinoma and Wilms tumor. In importantembodiments, cancers or tumors escaping immune recognition includeglioma, colon carcinoma, colorectal cancer, lymphoid cell-derivedleukemia, choriocarcinoma, and melanoma.

Dose and Administration

The IFN-alpha, as described herein, are administered in effectiveamounts. The effective amount will depend upon the mode ofadministration, the particular condition being treated and the desiredoutcome. It will also depend upon, as discussed above, the stage of thecondition, the age and physical condition of the subject, the nature ofconcurrent therapy, if any, and like factors well known to the medicalpractitioner. For therapeutic applications, it is that amount sufficientto achieve a medically desirable result.

The anti-cancer agent may be administered by any appropriate method.Dosage, treatment protocol, and routes of administration for anti-canceragents, including chemotherapeutic agents, radiotherapeutic agents,anti-cancer antibodies, immune checkpoint inhibitors, and anti-cancervaccines, are known in the art and/or within the ability of a skilledclinician to determine, based on the type of treatment, type of cancer,etc.

The length of time and modes of administration of IFN-alpha will vary,depending on the immune cells, type of tumor being treated, condition ofthe patient, and the like. Determination of such parameters is withinthe capability of the skilled clinician.

A variety of administration routes are available. The methods of theinvention, generally speaking, may be practiced using any mode ofadministration that is medically acceptable, meaning any mode thatproduces effective levels of the active compounds without causingclinically unacceptable adverse effects.

Modes of administration include oral, rectal, topical, nasal or mucosal,intradermal, or parenteral routes. The term “parenteral” includessubcutaneous, intravenous, intramuscular, or infusion. Intravenous orintramuscular routes are not particularly suitable for long-term therapyand prophylaxis. They could, however, be preferred in emergencysituations. In one embodiment, the IFN-alpha is administered orally, viavein injection, muscle injection, peritoneal injection, subcutaneousinjection, nasal or mucosal administration, or by inhalation via aninspirator.

EXAMPLES

The following examples are for illustrative purposes only and should notbe interpreted as limitations of the claimed invention. There are avariety of alternative techniques and procedures available to those ofskill in the art which would similarly permit one to successfullyperform the intended invention.

Example 1

NK cells are isolated from the mice using the EasySep™ Mouse NK CellIsolation Kit (STEMCELL™ Technologies) following the manufacturer'sprotocol. Isolated NK cells are incubated with the culture mediumcontaining IFN-alpha (Lee Biomolecular, San Diego, Calif.; 1×10³ U/1×10⁶cells) at 37° C. and 5% CO₂ for 7-30 days. The culture medium is changedevery 3 days with fresh IFN-alpha. Every 5 days, expanded NK cells aretransferred to T25 flask or T75 flasks at a concentration of 0.5×10⁶cells.

Nude mice are injected with HLA-negative tumor cells (subcutaneousinjection) to form a tumor that expresses no or reduced levels of HLA.

One to three days after the formation of tumors in the mice, the miceare injected via intravenous injection with 5×10⁶ NK cells that aretreated with IFN-alpha. The mice injected with the untreated NK cellsare used as control. Tumor growth in mice injected with treated NK cellsis delayed compared to mice injected with untreated NK cells.

Example 2

Nude mice are injected with HLA-negative tumor cells (subcutaneousinjection) to form a tumor that expresses no or reduced levels of HLA.

One to three days after the formation of tumor in the mice, the mice areinjected subcutaneously with control vehicle and IFN-alpha every threedays at 1×10⁴ U/m² for total 21 days. Tumor growth in mice injected withIFN-alpha is delayed compared to mice injected with control vehicle. Itcontemplated that the dosage of 1×10⁴ U/m² for IFN-alpha is below theminimal therapeutic amount that would lead IFN-alpha to have any directeffect on tumor growth.

What is claimed is:
 1. A method for treating cancer in a patient whosecancer is characterized by being HLA-negative or having reducedexpression, the method comprising administering to said patient anactivated NK cell population, wherein said activated population has beenactivated by an effective amount of IFN-alpha.
 2. A method for treatinga cancer, comprising: administering an immune cell to a subject in needthereof, wherein said immune cell is activated by IFN-alpha, wherein thecancer has no or reduced expression of a HLA compared to a normalcontrol.
 3. The method of claim 2, wherein the HLA comprises a class IHLA, a class II HLA, or a class III HLA.
 4. The method of claim 2,wherein a source of the immune cell is autologous, allogeneic, orxenographic.
 5. The method of claim 2, wherein the immune cell comprisesa T-cell, a B-cell, or a NK cell.
 6. The method of claim 2, wherein theimmune cell is administered intravenously, intraperitoneally,intramuscularly, or subcutaneously.
 7. The method of claim 2, whereinthe immune cell is activated by incubating the immune cell with theIFN-alpha ex vivo.
 8. The method of claim 7, wherein the immune cell isincubated ex vivo with the IFN-alpha at a concentration from 5×10¹U/1×10⁶ cells to 1×10⁴ U/1×10⁶ cells.
 9. The method of claim 2, whereinthe IFN-alpha comprises one or more of IFN-alpha 1, IFN-alpha 2,IFN-alpha 4, IFN-alpha 5, IFN-alpha 6, IFN-alpha 7, IFN-alpha 8,IFN-alpha 10, IFN-alpha 14, IFN-alpha 16, IFN-alpha 17, or IFN-alpha 21.10. The method of claim 9, wherein the IFN-alpha comprises IFN-alpha 2,IFN-alpha or IFN-alpha
 10. 11. The method of claim 2, wherein the immunecell is administered in combination with an anti-tumor agent.
 12. Themethod of claim 11, wherein the anti-tumor agent is selected from agroup consisting of a chemotherapy agent, a radiotherapy agent, ananti-cancer antibody, an immune checkpoint inhibitor, and an anti-cancervaccine.
 13. The method of claim 2, wherein the immune cell is furthermodified to express a tumor cell homing receptor on the outer cellsurface.
 14. The method of claim 13, wherein the tumor cell homingreceptor is a chimeric antigen receptor, an Fc receptor, or combinationsthereof.
 15. The method of claim 2, wherein the patient or subject ishuman
 16. A composition comprising immune cells and an effective amountof interferon alpha (IFN-alpha) to activate the immune cells.
 17. Thecomposition of claim 16, wherein the IFN-alpha comprises one or more ofIFN-alpha 1, IFN-alpha 2, IFN-alpha 4, IFN-alpha 5, IFN-alpha 6,IFN-alpha 7, IFN-alpha 8, IFN-alpha 10, IFN-alpha 14, IFN-alpha 16,IFN-alpha 17, or IFN-alpha
 21. 18. The composition of claim 16, whereinthe immune cells are NK cells.
 19. The composition of claim 18, whereinthe NK cells are NK-92 cells.
 20. The composition of claim 16,comprising IFN-alpha at a concentration from 5×10¹U/1×10⁶ cells to1×10⁴U/1×10⁶ cells.